Secondary Metabolites and Biological Activity of Invasive Macroalgae of Southern Europe

In this review a brief description of the invasive phenomena associated with algae and its consequences on the ecosystem are presented. Three examples of invasive algae of Southern Europe, belonging to Rodophyta, Chlorophyta, and Phaeophyta, were selected, and a brief description of each genus is presented. A full description of their secondary metabolites and biological activity is given and a summary of the biological activity of extracts is also included. In Asparagopsis we encounter mainly halogenated compounds. From Caulerpa, several terpenoids and alkaloids were isolated, while in Sargassum, meroterpenoids prevail.


Introduction
Alien species are plants, animals, or microbes that have been introduced and spread into new host regions, establishing populations that can become invasive if they interfere with the host ecosystem. These invasive species become established in natural or seminatural ecosystems, increasing in abundance and distribution and threatening biological diversity. They compete with native species, and usually have high reproductive rates assisted either by the lack of predators in the new environment or by the tolerance of a different range of environmental conditions. As a consequence, they are difficult to contain, harm biodiversity, and change the new host ecosystem [1].
Alien macroalgae are particularly likely to become invasive: their high reproductive rates, their production of toxic metabolites, and/or their perennial status make them more competitive than the native species, increasing the probability that they will become invasive. Several of these species periodically become a major problem, clogging waterways, fouling nets, and changing nutrient regimes in areas around fisheries, desalination facilities, and aquaculture systems [1]. They impact on local economies, such as fishery [2] and tourism.
The mechanism of invasion by macroalgae thus begins with transport (by means of fouling, ballast waters, or aquaculture), proceeds by establishment of the species (through biotic and abiotic factors), and ends with its spread and impact [3][4][5][6][7]. Management of this update problem requires adequate measures [8] and control procedures, such as mechanical means, biological control, and/or chemical remedies [9].
With global warming there is a general increase of the tendency of invasive episodes, this being a situation of concern especially for Southern Europe. The Mediterranean coast and Atlantic areas near Gibraltar are key points in the dynamics and spread of these phenomena. As an example, in 2016, several beaches in Gibraltar were interdicted by Dictyota invasions with direct impact on local near Gibraltar are key points in the dynamics and spread of these phenomena. As an example, in 2016, several beaches in Gibraltar were interdicted by Dictyota invasions with direct impact on local tourism, and remediation and management costs. However, macroalgae have underlying potential. Their commercial use as a source of nutraceuticals, food additives, biofuel, antifouling agents, or pharmaceuticals could be a way to exploit these phenomena in a more profitable way [4,10].
Thus, knowledge of the chemistry of these macroalgae is by no means out of date, as recent papers on the activity of algal extracts well document. Knowledge of their secondary metabolites and this review are also a starting point to the understanding of the chemistry of these species. There is a need, however, to fully characterize these invasive species in their new environment in order to make the most of their existence, and perform a strict correlation between metabolite and activity.
In this review we chose three genera of invasive species of the Mediterranean-Asparagopsis, Caulerpa, and Sargassum-as examples of the chemistry of red, green, and brown algae, respectively. Two of them-Asparagopsis and Caulerpa-are already signaled by the International Union for Conservation of Nature (IUCN) Centre for Mediterranean Cooperation [1].
The secondary metabolites of the chosen genus are presented and, when possible, the studied biological activities are given. Reference to their study as invasive specimens is also provided. A list of reports on the biological activity of extracts is also given. This review covers the literature up to 2017.

Structural Characterization and Biological Activity
In this paper a chemical and biological activity summary of three different genera of invasive species of Southern Europe is presented. The structural identification of the mentioned metabolites relies on the usual techniques such as NMR, IR, MS, and chemical transformations for the less recent publications. Although some of the studies include biological activities of the isolated metabolites, most of the papers only mention isolation and characterization.

Asparagopsis
Asparagopsis is a red seaweed genus of the family Bonnemaisoniaceae that has a diplohaplontic life cycle and a heteromorphic tetrasporophyte known as the "Falkenbergia" stage [11] Currently, only two species of this genus are accepted, A. armata and A. taxiformis, the former being endemic to the southern hemisphere and the latter being widely distributed in the tropics and subtropics [12]. Recently, a study of the lineages of this genus by DNA sequence was published [13].
Both species of this genus are native to Western Australia. A. armata is nowadays distributed throughout Europe in both the Atlantic and the Mediterranean basin, where it is highly invasive. A. taxiformis is invasive around the Indo-Pacific region, including Japan and Hawaii, and is currently widespread throughout the Mediterranean and along the Atlantic coast of Europe. While A. armata was probably introduced by maritime transport, A. taxiformis was probably introduced by oyster aquaculture [1].

Caulerpa
Green algae of the genus Caulerpa Lamouroux represent the single genus in the family Caulerpaceae, which consists of approximately 60 species worldwide, generally distributed in shallow-water tropical and subtropical marine habitats. One of its species, Caulerpa racemosa, also known as "sea grapes", is an edible marine green seaweed widely distributed throughout the South China Sea.
C. racemosa var. cylindrica is native to SouthWestern Australia, and is invasive in the Mediterranean [26][27][28] where its introduction is still speculative. Maritime traffic and aquarium trade are the most likely vectors. It can still be found in aquarium stores and is sold by internet retailers. C. taxifolia was accidentally introduced into the Mediterranean from a public aquarium in Monaco. Since then, it has spread rapidly due to its natural vegetative dispersal mechanism, its lack of natural grazers, and the ease of dispersion by boats, anchors, fishing nets, and aquaria [1].
From C. racemosa, fucosterol and the oxygenated sterols 7-10 in Figure 3 were isolated, together with both C-24 epimers of saringosterol 2 [30,31]. From C. racemosa, several varied metabolites were obtained by Yang et al. [31]. These include trans-phytol, trans-phytylacetate, α-tocopherolquinone, and the metabolites 11-17 in Figure 4. Ecotoxicological activities of 5 and 6 against a marine bioluminescent bacterium (Vibrio fischeri) were used as an assessment of their role in the environment, revealing high toxicities for both compounds (EC 50 effective concentration, 0.16 µM for 5 and 6). Additionally, both compounds were evaluated in antibacterial, antifungal, and cytotoxicity assays. Compounds 5 and 6 exhibited mild antibacterial activities against the human pathogen Acinetobacter baumannii.

Caulerpa
Green algae of the genus Caulerpa Lamouroux represent the single genus in the family Caulerpaceae, which consists of approximately 60 species worldwide, generally distributed in shallow-water tropical and subtropical marine habitats. One of its species, Caulerpa racemosa, also known as "sea grapes", is an edible marine green seaweed widely distributed throughout the South China Sea.
C. racemosa var. cylindrica is native to SouthWestern Australia, and is invasive in the Mediterranean [26][27][28] where its introduction is still speculative. Maritime traffic and aquarium trade are the most likely vectors. It can still be found in aquarium stores and is sold by internet retailers. C. taxifolia was accidentally introduced into the Mediterranean from a public aquarium in Monaco. Since then, it has spread rapidly due to its natural vegetative dispersal mechanism, its lack of natural grazers, and the ease of dispersion by boats, anchors, fishing nets, and aquaria [1].
From C. racemosa, fucosterol and the oxygenated sterols 7-10 in Figure 3 were isolated, together with both C-24 epimers of saringosterol 2 [30,31].  Ecotoxicological activities of 5 and 6 against a marine bioluminescent bacterium (Vibrio fischeri) were used as an assessment of their role in the environment, revealing high toxicities for both compounds (EC50 effective concentration, 0.16 μM for 5 and 6). Additionally, both compounds were evaluated in antibacterial, antifungal, and cytotoxicity assays. Compounds 5 and 6 exhibited mild antibacterial activities against the human pathogen Acinetobacter baumannii.

Caulerpa
Green algae of the genus Caulerpa Lamouroux represent the single genus in the family Caulerpaceae, which consists of approximately 60 species worldwide, generally distributed in shallow-water tropical and subtropical marine habitats. One of its species, Caulerpa racemosa, also known as "sea grapes", is an edible marine green seaweed widely distributed throughout the South China Sea.
C. racemosa var. cylindrica is native to SouthWestern Australia, and is invasive in the Mediterranean [26][27][28] where its introduction is still speculative. Maritime traffic and aquarium trade are the most likely vectors. It can still be found in aquarium stores and is sold by internet retailers. C. taxifolia was accidentally introduced into the Mediterranean from a public aquarium in Monaco. Since then, it has spread rapidly due to its natural vegetative dispersal mechanism, its lack of natural grazers, and the ease of dispersion by boats, anchors, fishing nets, and aquaria [1].

Mar. Drugs 2018, 16, x FOR PEER REVIEW 5 of 28
From C. racemosa we can also find two prenylated p-xylenes [32] 18 and 19 and racemosins A 20 and B 21 [33] ( Figure 5). From C. prolifera [34], caulerpin 22 was isolated ( Figure 6). In in vitro bioassays, the compounds 18 and 19 exhibited a broad spectrum of antifungal activity against Candida glabrata, Trichophyton rubrum, and Cryptococcus neoformans with MIC80 (minimum inhibitory concentration) values between 4 and 64 μg/mL when compared to amphotericin B (MIC80 values of 2.0, 1.0, and 4.0 μg/mL, respectively) as a positive control and showed no growth inhibition activity against the tumor cells HL60 and A549 [32].
From C. bikinensis, compounds 30-32 were isolated and tested as feeding deterrents [38]. The diacetate 30 and the dialdehyde 31 were found to be toxic to the Pacific damselfish Pomacentrus phillipinus at the 10 and 5 µg/mL levels. Feeding deterrence effects were reliably produced from 30 and 31 when tested at 1000 ppm levels against similar herbivorous fishes. The cytotoxicities of these compounds against the fertilized egg of the Pacific sea urchin Lytechinus pinctus were also measured. Again, 30 and 31 showed ED 50 (effective dose) values of 2 and 1 µg/mL. The activities noted for these metabolites reinforce their likely roles in nature as agents of chemical defense.
From C. prolifera, 25 was isolated and its absolute configuration determined as S [37].
A study of C. taxifolia from Cap Martin, Côte d'Azur, at the time considered an invasive species, allowed the isolation of compounds 24-28, for which no absolute configurations were determined. The proposed configurations were based on biosynthetic considerations [36].
From a larger study on algae of the order Caulerpales, diterpene 43 was isolated from C. brownii. [41]. Compound 43 had already been tested for biological activities. It showed antibacterial activity towards the pathogenic bacteria Staphylococcus aureus and Bacilus subtilis. It was also tested against marine bacteria and was found to be inhibitory towards Vibrio harveyi and V. leiognathi. It is also active against E. coli and V. anguillarum [41]. Handley reported the isolation of diterpenes 41-54 from branched and unbranched specimens of C. brownii and compound 50 was reported for the first time as a natural product [42].
From C. trifaria, diterpene 40 was isolated and the depicted configuration is proposed [40].

Sargassum
Sargassum is a genus of brown seaweeds with tropical and subtropical distribution, existing in all oceans. It is a large genus, comprising over 350 species. Some of its species are used in food in Japan and Korea, such as S. fusiforme and S. muticum. Due to air vesicles, S. natans and S. fluitans form large floating masses. S. muticum is invasive in the Mediterranean [43,44] and in Western Europe [45], and seems to have been introduced by the business of oyster culture [46].
A recent review on the therapeutic potential and health benefits of these species has been published [47].
The data showed that all the steroids exhibited activities causing morphological abnormality of P. oryzae mycelia. Fucosterol and 24-ethylcholesta-4,24(28)-dien-3,6-dione exhibited significant cytotoxicity toward P388 cancer cells, whereas 61 and 56 showed mild activity against the growth of HL-60 cancer cells. In the antitumor screen using a panel of human cell lines only the epoxy sterol 10 showed some cytotoxicity against several human cell lines. Compounds 62, 9, and 10 were also evaluated for HIV (Human immunodeficiency virus) growth inhibition activity in H9 lymphocytes. The EC 50 and IC 50 values for 9 were 0.500 and 0.975 mg/mL, whereas 62 and 10 were inactive.
From the genus Sargassum we can also find reports on the isolation of quinones and hydroquinones, chromenes, and varied structures.
From the genus Sargassum we can also find reports on the isolation of quinones and hydroquinones, chromenes, and varied structures.
From S. fallax [55], 67-71 were isolated. Sargaquinone 67 was isolated as a mixture with sargaquinoic acid 68. Both 68 and 69 were found to display moderate antitumor activity when tested against P388 cells. They displayed only weak activity against Bacillus subtilis.
From S. fallax [55], 67-71 were isolated. Sargaquinone 67 was isolated as a mixture with sargaquinoic acid 68. Both 68 and 69 were found to display moderate antitumor activity when tested against P388 cells. They displayed only weak activity against Bacillus subtilis.
From S. herophyllum [56], 67, 69, and 72 were isolated. They displayed moderate antiplasmodial activity against P. falciparum.  [57], 73-76 were isolated. Compounds 74-76 displayed strong antioxidant activity, such as an inhibitory effect on NADPH-dependent lipid peroxidation in rat liver microsomes and radical-scavenging effect on DPPH (1,1-diphenyl-2-picrylhydrazyl). The inhibitory effect on lipid peroxidation was shown to be the same or stronger than that of the positive control, α-tocopherol. The authors identify the absence or presence of an unsaturated cis carbon-carbon double bond in the long-chain fatty acid ester moiety of 75 and 76 as responsible for the large difference in the inhibitory activity. Both compounds were found to have moderate radical-reducing effect on DPPH at a dose of each sample of 100 mg/mL. Based on these preliminary results, the author suggest that the hydroquinone moiety of 74 must participate in antioxidant activity, while in compounds 75 and 76, hydrolysis of their ester group occurs first, and the resulting 74 may owe this activity. Antiproliferative activity of 74-76 against Colon 26-L5 cell was also evaluated. Compounds 74 and 76 showed relatively strong cytotoxic activity while moderate activity in the case of 75 was observed.
From S. paradoxum [58], 67-71 together with 77-83 were identified by HPLC-NMR and HPLC-MS. Some of the compounds were isolated by bioguided fractioning and tested for their biological activity. Compared to the antibiotic ampicillin, the isolated compounds were far less potent against S. aureus and S. pyogenes. However, compounds 69, 71, 80, and 260 were more potent against P. aeruginosa than ampicillin. There was no difference in activity between compounds with the hydroquinone or the p-benzoquinone moieties. The activity observed for sargaquinone 67, the simplest of the meroditerpenoids isolated, suggests that the unsubstituted meroditerpenoid skeleton is responsible for the activity against P. aeruginosa. The addition of an alcohol group at position 12 or 20 (70, 77, 78, 82, and 83) appears to reduce the activity against P. aeruginosa, but increases the activity against S. pyogenes. Finally, incorporation of a carboxylic acid at position C-20 (69 and 68) gives rise to activity against S. aureus and S. aureus MRSA Methicillin-resistant Staphylococcus aureus).
From S. thunbergii [62,63], sargaquinoic acid 68 and sargahydroquinoic acid 69 were isolated. Since S. thunbergii was shown to inhibit adipogenesis in pre-adipocytes while enhancing osteoblast differentiation of pre-osteoblasts, and 68 and 69 were isolated in a bioguided study, the authors suggest that these two compounds possess osteoblastogenesis-enhancing abilities [63].
From S. tortile [66], 67 and 89-95 were isolated. Compounds 68 and 69 were also isolated from S. yezoense [67]. Their effect on the transcriptional activity of PPARs (Peroxisome proliferator-activated receptors) was studied. The authors suggest that both compounds could be possible candidates for the treatment of type-2 diabetes and dyslipidemia. From S. yezoense [68], 85-88 were also isolated. Their antidiabetic potential was also evaluated.
Mar. Drugs 2018, 16, x FOR PEER REVIEW 13 of 28 both compounds could be possible candidates for the treatment of type-2 diabetes and dyslipidemia. From S. yezoense [68], 85-88 were also isolated. Their antidiabetic potential was also evaluated.
From S. sagamianum, the isolation of 125 and its proapoptotic activity is described [65]. Its anticholinesterase activity and potential use in Alzheimer's disease is also described [64].
From S. siliquastrum, Yoon [70] reported the isolation of 100, and its potential as a novel antiinflammatory agent was investigated. Lee [71] reported the isolation of 101-106. The antioxidant activity of these compounds was evaluated by various antioxidant tests, such as scavenging effects on generation of intracellular ROS (reactive oxygen species), increments of GSH (glutathione) level, and inhibitory effects on lipid peroxidation in human fibrosarcoma HT 1080 cells. Compounds 101-106 significantly decreased generation of intracellular ROS and inhibited lipid peroxidation while they increased levels of intracellular GSH at a concentration of 5 μg/mL. Compound 101 was also isolated by Heo [72] and its anti-inflammatory activity against lipopolysaccharide-exposed RAW 264.7 cells was evaluated. Jang [73] reported the isolation of 101 and 102, together with 107-120. Although the configurations of 101, 102, and 120 are relative, for 109-115 the absolute configurations of the hydroxyl groups were determined by a Mosher's method. Using DPPA (1,1-diphenyl-2picrylhydrazyl), all of the compounds exhibited significant radical-scavenging activity in the range of 87-91% at the concentration of 100 μg/mL. In addition, compounds 111 and 117 displayed 82.7 and 80.0% inhibition, respectively, toward butylcholine esterase at the same concentration, while the other sargachromanols showed weaker or negligible activity. Cho reported the isolation of 127 and its antioxidant activity [77].
From S. sagamianum, the isolation of 125 and its proapoptotic activity is described [65]. Its anticholinesterase activity and potential use in Alzheimer's disease is also described [64].
From S. siliquastrum, Yoon [70] reported the isolation of 100, and its potential as a novel anti-inflammatory agent was investigated. Lee [71] reported the isolation of 101-106. The antioxidant activity of these compounds was evaluated by various antioxidant tests, such as scavenging effects on generation of intracellular ROS (reactive oxygen species), increments of GSH (glutathione) level, and inhibitory effects on lipid peroxidation in human fibrosarcoma HT 1080 cells. Compounds 101-106 significantly decreased generation of intracellular ROS and inhibited lipid peroxidation while they increased levels of intracellular GSH at a concentration of 5 µg/mL. Compound 101 was also isolated by Heo [72] and its anti-inflammatory activity against lipopolysaccharide-exposed RAW 264.7 cells was evaluated. Jang [73] reported the isolation of 101 and 102, together with 107-120. Although the configurations of 101, 102, and 120 are relative, for 109-115 the absolute configurations of the hydroxyl groups were determined by a Mosher's method. Using DPPA (1,1-diphenyl-2-picrylhydrazyl), all of the compounds exhibited significant radical-scavenging activity in the range of 87-91% at the concentration of 100 µg/mL. In addition, compounds 111 and 117 displayed 82.7 and 80.0% inhibition, respectively, toward butylcholine esterase at the same concentration, while the other sargachromanols showed weaker or negligible activity. Cho reported the isolation of 127 and its antioxidant activity [77].
From S. tortile, Kato [74] reported the isolation of 123 and 124, together with their activity as attractants of the swimming larvae of Coryne uchidai. Kikuchi [75,76] reported the isolation and identification of 126. Absolute configurations were determined by ECD (electronic circular dichroism).
From S. fusiformis [79], fucoxanthine 140 was isolated by microwave-assisted extraction coupled with high-speed countercurrent chromatography. This compound was also isolated from S. elegans [54] and S. heterophyllum [56]. The antioxidant potential of 140 was evaluated [54] and it also showed a moderate antiplasmodial activity (IC 50 = 1.5 µm) [56]. In order to assess the selectivity of fucoxanthin 140 for P. falciparum, the toxicity against a Chinese hamster ovarian cell line was evaluated. The relatively low cytotoxicity of fucoxanthin (IC 50 = 83.7 µm) translated into a promising selectivity index (SI = antiplasmodial IC 50 /cytotoxicity IC 50 ) of 54 [56]. From S. Kjellmanium, 141 [80] and 142 [81] were isolated. For both compounds, the structure was confirmed by single-crystal X-ray analysis.
From S. siliquastrum [61], compounds 143-159 were isolated. They showed moderate to significant radical-scavenging activity in DPPH assays. The 100-fold increase in radical-scavenging activity of the diphenolic isonahocols relative to the monophenolic nahocols indicated the role of the phenolic group in this activity. None of these compounds exhibited antimicrobial activity against Gram-positive or -negative bacteria or against pathogenic fungi. Conversely, the isonahocols 154-159 showed slight activity against sortase A derived from Staphylococcus aureus. The nahocols 143-153 showed no inhibitory activity against sortase A. These compounds were, however, weakly active against isocitrate lyase derived from Candida albicans.
Finally, we can also find reports on the antifouling activity of fats and phthalic acid derivatives from S. confusum [83] and the isolation of farnesylacetones from S.micracanthum [84,85], from S. sagamianum with moderate anticholinesterase activity [86], and from S. siliquastrum with a moderate vasodilatation effect on the basilar arteries of rabbits [87]. Three linear bisnorditerpenes were also isolated from unidentified Sargassum sp. [88].

Biological Activity of Extracts
Macroalgae continue to attract the attention of researchers, as several reports on the activity of extracts in the literature testify. From the chosen genera here mentioned the following reports can be found.

Conclusions
It is interesting to find the differences between the chemical compositions of all three genera. Asparagopsis is mainly rich in halogenated compounds, Caulerpa shows metabolites from varied biosynthetic routes, and Sargassum is rich in meroterpenoids. While biological activity of Asparagopsis metabolites is scarce, Caulerpa metabolites were shown to have inhibitory activity of PTPs, and to be neuroprotective, deterrents, and antibacterial. Sargassum metabolites are cytotoxic to cancer cells, and are antiplasmodial and antioxidants. Of course, only the more recent literature mentions biological activity results for the isolated metabolites. Extracts from all three genera show varied biological activities that make this a promising area of research. There is, however, a need to reinvestigate these genera as particular invasive species in their new host habitat since almost no reports are found on their chemistry. Their success in new environments can surely be correlated to their secondary metabolism and could provide new uses for otherwise noxious species.
Author Contributions: The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. All authors contributed equally.
Funding: This research received no external funding.
Acknowledgments: Authors acknowledge the Associated Laboratory for Sustainable Chemistry-Clean Processes and Technologies LAQV, which is financed by national funds from FCT/MEC(UID/QUI/50006/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007265) for ensuring the conditions and means to carry out this work.

Conflicts of Interest:
The authors declare no conflicts of interest.